[unreadable] Recently, we have succeeded in the reduction of mutant mtDNA content in cells carrying heteroplasmic T8993G mutation, which causes the NARP and MILS syndromes, heritable fatal mitochondrial disorders. The rates of ATP production and oxygen consumption in treated cells improved, while rates of lactate secretion decresed. This fact indicates the functional restoration of electron transfer chain. Our approach, if used therapeutically, has a potential of becoming the first therapy capable of reversing the symptoms for any mitochondrial disorder. However, the novelty of this approach as therapeutic modality makes it imperative to investigate its safety. Ideally, this would be done in experimental animals. Unfortunately, currently there is no animal model for either T8993G NARP, or (with perhaps one exception) any other human disease caused by the mutation in mtDNA. Recently, there was an important breakthrough: several groups demonstrated their ability to generate transmitochondrial mice carrying mutation, which was induced in tissue culture by selecting cells for resistance to antibiotic chloramphenicol. Therefore, if we had cultured mouse cells carrying mutations synonymous to those found in human diseases, we could generate mouse models for these diseases. However, neither such cells, nor methods to generate such cells exist today. Therefore, the goal of this application is to develop a technology for the generation of a collection of mouse cell lines with various mutations in mtDNA, to generate such a collection, and to do an initial characterization of this collection. We plan to first generate random mutations in mouse mtDNA by overexpressing the error-prone mitochondrial DNA-polymerase gamma and then to force cells with random mtDNA mutations through the bottleneck by first depleting and then repleating mtDNA in these cells. If successful, proposed studies will provide a significant breakthrough as they will not only help us to move forward with our NARP studies, but will also dramatically accelerate the search for effective therapies by making animal models for mtDNA diseases routinely available. [unreadable] [unreadable] [unreadable]